Immunocytochemistry of the rat brain revealed that chymotrypsin (CT) cell bodies were present in nucleus basalis (nucleus of the tractus diagonalis). Studies with the cholinergic nerve marker choline acetyltransferase (ChAT) revealed that the CT cells were in close proximity to the ChAT-containing cells but did not colocalize. In the cortex, neuronal cells and varicose fibers were infrequently observed to contain CT-like immunoreactivity. The human cortex and hippocampus were similarly observed to contain positive-staining cells and plexuses of varicose nerve fibers. In human Alzheimer brains, preliminary observations revealed CT immunopositive neuronal cells, processes, and punctate material in what appears to be microglia. Positive stain was observed in the nuclei of cortical neurons and cerebellar granule cells of normal and Alzheimer's brains. CT-like staining was observed in close proximity to the placques (thioflavin positive) of Alzheimer's brains. The presence of CT in brain microglia suggests a possible role of protease activity following phagocytosis. Using the RNase protection procedure developed in our laboratory for quantitation of mRNA in discrete micropunches of brain regions, we have detected CT mRNA in rat brain. We found that regions such as the hippocampus, striatum, thalamus and cortex contain CT mRNA. Further evidence that CT mRNA exists in rat brain regions was provided by Northern blots. The presence of CT in the brain is of particular interest in aging and neurodegenerative diseases such as Alzheimer's disease. A variety of peripheral organs (intestinal tract, heart, lung, kidney, liver, spleen, thymus, adrenal, bladder, testis, ovary and pancreas) were also studied. The presence of CT mRNA was detected in the pancreas, stomach, duodenum, jejunum, ovary and spleen. Northern blot analysis of mRNA revealed a CT message in the stomach, jejunum, spleen, ovary and pancreas. Immunofluorescence microscopy with polyclonal and monoclonal antibodies to human CT detected (a) CT-like immunoreactivity in endocrine-like cells of the basal third of the villi of the stomach; (b) positive immunoreactivity in some epithelial cells of the duodenum and jejunum, rare cells were seen in the ileum; (c) positive immunoreactivity in the acini of the exocrine glands of the pancreas. The presence of CT in neuroendocrine-like cells of the stomach suggests the possibility that CT may serve to cleave peptides from their storage sites in secretory granules.